Recent advances in biotechnology and chemistry have made available an increasing number of potential therapeutic agents. However, many agents fail at the preclinical or early clinical stage due to poor pharmacokinetics. Other potential targets for pharmaceutical agents are largely ignored due to anticipated problems of pharmaceutical agent delivery. Oral delivery of compounds is advantageous for reducing treatment costs and increasing patient acceptance. However, formulating compounds for efficient oral bioavailability has proven particularly difficult because of problems associated with uptake and susceptibility to metabolic enzymes in the intestinal tract. Likewise, delivery of compounds across the blood brain barrier or targeting compounds to specific tissues has proven problematic.
There are two major specific transport systems of xenobiotics into and through cells: carrier-mediated systems and receptor-mediated systems. Some xenobiotics can also be taken up by passive diffusion between or through cells. Carrier-mediated systems are effected by transport proteins that are anchored to the cell membrane, typically by a plurality of membrane-spanning loops and function by transporting their substrates via an energy-dependent flip-flop mechanism. Carrier-mediated transport systems are involved in the active transport of many important nutrients such as vitamins, sugars, and amino acids, as well as xenobiotic compounds. The carrier systems are involved in transport of such molecules from the lumen of the intestine into the systemic circulation or across the blood brain barrier. Carrier-mediated transporters are also present in organs such as liver and kidney, where the proteins are involved in the excretion or reabsorption of circulating compounds.
Receptor-mediated transport systems differ from the carrier-mediated systems in that rather than ferrying the substrate/ligand across the membrane, substrate binding triggers an invagination and encapsulation process that results in the formation of various transport vesicles to carry the substrate (and sometimes other molecules) into and through the cell. This process of membrane deformations that result in the internalization of certain substrates and their subsequent targeting to certain locations in the cytoplasm is referred to as endocytosis. Endocytosis encompasses several specific variations, including, for example, receptor mediated endocytosis (RME).
RME involves several defined steps beginning with the binding of a substrate to a cell-surface receptor and subsequent invagination of the membrane to form an internal vesicle variously called an early endosome, a receptosome or CURL (compartment of uncoupling receptor and ligand). In some endocytic events, after a substrate binds to its specific receptor, the substrate-receptor complex accumulates in coated pits that contain high concentrations of clathrin subunits that appear to aid in the membrane invagination process. Following internalization, the clathrin coat is lost and the pH in the endosome is lowered, thus resulting in the dissociation of the receptor-substrate complex. The endosome moves randomly or along microtubules to the trans-Golgi reticulum where the endosome is converted into one of a variety of different sorting vesicles (e.g., tubulovesicular complexes and late endosomes or multivesicular bodies). The fate of the receptor and substrate depends upon the type of sorting vesicle formed. Some ligands and receptors are recycled to the cell surface where the substrate is released and the receptor reinternalized into the membrane. In other instances, the substrate is directed to and destroyed in a lysosome, and the receptor is recycled. One type of RME is transcytosis, which refers to the process wherein an endocytotic vesicle is transported to the opposite membrane surface of a polarized cell.
RME is capable of transporting a variety of compounds, including immunoglobulins, lectins, vitamins and metal ions. Although some conflicting data has been obtained, it has been reported that certain small particles such as nanoparticles can be transported by endocytotic routes, particularly through both enterocytes and M-cells located in the intestine. Nanoparticles can be made of a variety of materials, and are typically between 1-999 nm in diameter.
A number of attempts have been made to assay or identify substrates of various transport proteins or to modify pharmaceutical agents to be improved substrates of transport proteins. (See, e.g., Kramer et al, J. Biol. Chem. (1994), 269:10621; Mills et al, Biochim. Biophys. Acta (1992), 1126: 35; Börner et al, Eur. J. Biochem. (1998) 255: 698; Dieck et al, Glia, (1999) 25: 10; Otto et al, Am. J. Physiol. (1996) 271:C210; Abe et al, Biooconjugate Chem. (1999) 10: 24); Hussain et al, Pharm. Res. 1997, 14, 613 and McClean et al, Eur. J. Pharm. Sci. 1998, 6, 153). Some assays have involved measuring the uptake or transcellular flux of labeled compounds. Some assays have involved measuring uptake of unlabelled compound by HPLC. There have been a few successes. For example, Valtrex, a derivative of Zovirax, has 54% oral availability compared with Zovirax's 10-20% oral bioavailability. However, existing assays are tedious, low throughput, and the delivery of many pharmaceutical agents and potential pharmaceutical agents remains to be improved.